Peaks studio 8

A. lumbricoides extract (1000 µg/mL) was digested using the ProteoExtract All-in-One Trypsin Digestion Kit (EMD Millipore, Billerica, MA, USA) and desalted using C18ZipTips (EMD Millipore, Billerica, MA, USA). Resulting peptides were separated by reverse-phase nano-high performance liquid chromatography (HPLC, Dionex Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany, column: PepSwift Monolithic Nano Column, 100 µM × 25 cm, Dionex). The column was developed with an acetonitrile gradient (Solvent A: 0.1% (v/v) FA/0.01% (v/v) TFA/5% (v/v) ACN; solvent B: 0.1% (v/v) FA/0.01% (v/v) TFA/90% (v/v) ACN; 5–45% B in 60 min) at a flow rate of 1 µL/min at 55 °C. The HPLC was directly coupled via nano electrospray to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). Capillary voltage was 2 kV. For peptide assignment, a top 12 MS/MS method was used with the normalized fragmentation energy set to 27%. The protein was identified with PEAKS Studio 8 (Bioinformatics Solutions, Waterloo, ON, Canada) using the UniProt (SwissProt/TrEMBL) sequence database. Only peptides with high confidence scores (−10lgp ≥ 35, corresponding to false discovery rate (FDR) < 0.5%) were considered in the database searches [18 (link)]. Abundance of Al-CPI in the extract was calculated based on the “Area” parameter from the summarized proteomic search results [23 (link)].

All MS/MS ion spectra were analyzed using Peaks Studio 8.0 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada) for processing, de novo sequencing and database searching. Resulting sequences were searched against the UniProt Human Proteome database (downloaded 5 May 2018) with mass error tolerances of 10 ppm and 0.02 Da for parent and fragment, respectively. The digestion enzyme trypsin allowed for two missed tryptic cleavages, Carbamidomethyl of cysteine specified as a fixed modification, and Oxidation of methionine and acetyl of the N-terminus as variable modifications. FDR estimation was enabled. Peptides were filtered for −10log p ≥ 20, and proteins were filtered for −10log p ≥ 15 and one unique peptide. For all experiments, this gave an FDR of <1% at the peptide-spectrum match level. Proteins sharing significant peptide evidence were grouped into clusters.

In vitro kinase assays of GST‐tagged AKT1 and Flag‐tagged SOD2 WT or S27A were done, unlabeled ATP was used in the reactions. Then the reactions were terminated by freezing and processed for mass spectrometry. In brief, proteins were precipitated with acetone and the protein pellets were dried by using a SpeedVac for 1–2 min. The pellets were subsequently dissolved in 8 m urea, 100 mm tris‐HCl, pH 8.5. 5 mm TCEP (Thermo Scientific), and 10 mm iodoacetamide (Sigma) and alkylation were added to the solution and incubated at room temperature for 30 min, respectively. The protein mixtures were diluted four times and digested overnight with Trypsin at 1:50 w/w (Promega). The digested peptide solutions were desalted using a MonoSpin C18 column (GL Science, Tokyo, Japan) and dried with a SpeedVac.
The peptide mixtures were analyzed by LC/tandem MS (MS/MS). Data‐dependent tandem mass spectrometry (MS/MS) analysis was performed with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA). The acquired MS/MS data were analyzed against a Swiss‐Prot Homo sapiens database using PEAKS Studio 8.5 (Bioinformatics Solutions, Waterloo, Ontario, Canada). The database search parameters were set as the followings: MS and MS/MS tolerance of 20 ppm and 0.1 Da, respectively, FDR was set as 1% and protein identification threshold was set as (−10 logP) ≧ 20.

Following LC-MS/MS analysis, the QTOF data were searched using the Peaks Studio 8.5 search algorithm (Bioinformatics Solutions, Waterloo, ON, Canada) against an in-house Mytilus sequence database and the uniprot-mollusca-200718-290-109 sequence database. The following parameters were used: A precursor mass tolerance of 0.15 Da and a fragment mass tolerance of 0.15 Da were allowed, trypsin was specified as the digestive enzyme, and up to 2 missed cleavages were allowed. Carbamidomethyl (C) was specified as a fixed modification, and oxidation (M) and deamidation (NQ) were chosen as variable modifications. A false discovery rate (FDR) of 1% was used for peptide identification in Peaks. In addition, the Peptide Hit Threshold (−10log_) was set to 30, de novo only 15% of ALC, and only proteins with a minimum of 1 unique peptide identification were included.

The proteomics analysis was done in collaboration with the Biomolecular/Proteomics Mass Spectrometry Facility at UCSD as described (Shevchenko, Wilm, Vorm, & Mann, 1996). Briefly, proteins were subjected to in‐gel tryptic digestion and the peptides were analyzed by ultra‐high‐pressure liquid chromatography (UPLC) coupled with tandem mass spectroscopy (LC‐MS/MS) using nanospray ionization. The nanospray ionization experiments were performed using an Orbitrap fusion Lumos hybrid mass spectrometer (Thermo) interfaced with nanoscale reversed‐phase UPLC (Thermo Dionex UltiMate™ 3000 RSLC nano System). Protein identification and label‐free quantification were carried out using PEAKS Studio 8.5 (Bioinformatics Solutions Inc.)